Squamous cell carcinomas of the skin (SCC) are a leading cause of death in organ transplant recipients and treatment of non-melanoma skin cancers, of which SCC is the second most frequent type, account for 4.5% of all Medicare cancer costs. Immune evasion in human SCC primarily results from aberrant T cell homing. Vessels in SCC lack expression of the critical cutaneous T cell homing addressin E-selectin and exclude the population of antigen-experienced skin homing T cells most capable of recognizing the tumor. Treatment of SCC with the TLR7 agonist imiquimod induces vascular E-selectin, influx of skin homing effector T cells and immunologic destruction of SCC, but associated broad-spectrum immune activation makes these medications less useful in solid organ transplant recipients in whom there is concern about engendering graft rejection. There is therefore a need to identify novel agents that can induce endothelial activation and restore appropriate T cell homing without broad, nonspecific activation of the immune system. A validated and reproducible screen to identify small molecules that induce endothelial cell E-selectin expression is described in this proposal. In the primary screen, HUVEC are grown to confluence, exposed to test compounds and immunostained for E- selectin expression. Prior similar screens have been performed to identify agents that inhibit or potentiate TNF1-induced endothelial activation but this is the first screen to search for independently acting small molecule inducers of endothelial activation. Secondary screens are in place to eliminate compounds with intrinsic optical interference and to validate findings using a second independent methodology, quantitative RT- PCR. Tertiary screens are in place to confirm activity of prioritized hit compounds on human dermal microvascular endothelial cells and to separate out compounds that do not induce dendritic cell activation, a hallmark of broad-spectrum immunologic activation. Additional biologic and preclinical screens are in place to confirm that candidate molecules function in the presence of nitric oxide, penetrate human skin, act within the SCC tumor microenvironment to induce E-selectin in vitro and enhance T cell homing in mice xenografted with human SCC tumors. We envision two classes of probes arising from these studies: one class that does not induce dendritic cell maturation and will be useful in the topical treatment of skin cancers in organ transplant recipients and a second class of compounds that induce activation of both dendritic cells and endothelial cells and would be useful in the treatment of skin cancers in non-transplanted patients and for the treatment of other internal human malignancies.